Protein quantitation device

ABSTRACT

Systems for protein quantitation using a Fabry-Perot interferometer. In one arrangement, a quantitation device includes an infrared source, a sample holder, and a Fabry-Perot interferometer positioned to receive infrared radiation from the source passing through a sample on the sample holder. A band pass optical filter sets the working range of the interferometer, and radiation exiting the interferometer falls on a detector that produces a signal indicating the intensity of the received radiation. A controller causes the interferometer to be tuned to a number of different resonance wavelengths and receives the intensity signals, for determination of an absorbance spectrum.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/569,065 filed Oct. 6, 2017 and titled “ProteinQuantitation Device”, the entire disclosure of which is herebyincorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

The measurement of the concentration or quantity of a protein or otheranalyte in a mixture is an integral part of much biochemical andbiomedical research. For example, it may be desired to quantify theresults of a protein purification procedure, or it may be desired tomeasure the amount of DNA or RNA in a mixture. Protein quantitation mayalso be used in disease diagnosis, and other applications.

Various techniques and devices have been developed to perform suchquantitation studies. For example, the Bradford assay is a colorimetricassay in which Coomassie stain becomes blue in contact with proteins.The optical density (absorbance) of the mixture is measured with aspectrophotometer, and the result compared with a standard curve.

Other techniques exploit the fact that many analytes, includingproteins, absorb light at specific wavelengths. For example, onetechnique is to illuminate a drop of the mixture, separate the lightexiting the drop in to its spectral components, and measure thecomponents with a series of detectors. The resulting spectrum can beexamined to note the amount of light that was absorbed at wavelengths ofinterest.

Another technique often used to quantitate proteins and other analytesis Fourier transform infrared (FTIR) spectroscopy. In this technique, asample is illuminated with broadband infrared light in a Michelsoninterferometer. As the interferometer is adjusted, certain wavelengthsare filtered out by destructive interference within the interferometer.The resulting interferogram is processed using Fourier transform methodsto extract the absorbance spectrum of the mixture.

Prior methods of quantitation have been time consuming, or involvedbulky and expensive equipment.

BRIEF SUMMARY OF THE INVENTION

According to one aspect, a device for measuring absorption of light by asample comprises a source of infrared radiation, a sample holderpositioned to place a sample in a location to receive infrared radiationfrom the source, and a Fabry-Perot interferometer positioned to receiveinfrared radiation originating from the source and passing through thesample. The Fabry-Perot interferometer comprises a pair of spaced-apartreflective surfaces, at least one of which is movable to change thespacing between the reflective surfaces. The device further comprises adetector positioned to receive infrared radiation from the Fabry-Perotinterferometer and to produce an output signal indicating the intensityof the infrared radiation received at the detector. The device furtherincludes a band pass optical filter positioned in an optical path of thedevice such that infrared radiation from the source passes through theoptical filter before reaching the detector. The device also includes acontroller coupled to the source, the Fabry-Perot interferometer, andthe detector. The controller includes a processor programmed to causethe Fabry-Perot interferometer to be tuned to a series of differentresonant wavelengths, receive the output signal of the detector at eachof the series of resonant wavelengths, and record an absorbance spectrumindicating the absorbance of the sample as a function of infraredradiation wavelength. In some embodiments, the source of infraredradiation is a micromachined resistive source. In some embodiments, thesource of infrared radiation is hermetically sealed. In someembodiments, the Fabry-Perot interferometer is a micromachinedFabry-Perot interferometer. In some embodiments, the device furthercomprises an optical system positioned to direct infrared radiationpassing through the sample toward an entrance aperture of theFabry-Perot interferometer. In some embodiments, the detector is apyroelectric detector. In some embodiments, the source is modulated at apredetermined measurement frequency. In some embodiments, the devicefurther comprises a lock-in amplifier that receives the signal from thedetector, enabling lock-in detection of the infrared radiationintensity. In some embodiments, the band pass optical filter isimplemented using a coating on an optical element of the device. In someembodiments, the band pass optical filter is a first band pass opticalfilter and passes infrared radiation in a first wavelength band, and thedevice further comprises: a second band pass optical filter that passesinfrared radiation in a second wavelength band different from the firstwavelength band; and a mechanism for removing the first optical filterfrom the optical path and placing the second filter in the optical pathsuch that infrared radiation from the source passes through the secondoptical filter before reaching the detector. In some embodiments, theseries of wavelengths is a first series of wavelengths and theabsorbance spectrum is a first absorbance spectrum, and the processor ofthe controller is further programmed to: cause the Fabry-Perotinterferometer to be tuned to a second series of different resonantwavelengths with the second band pass optical filter in place in theoptical path; receive the output signal of the detector at each of thesecond series of resonant wavelengths; and record a second absorbancespectrum indicating the absorbance of the sample as a function ofinfrared radiation wavelength within the range of wavelengths passed bythe second band pass optical filter. In some embodiments, the processorof the controller is further programmed to combine the first and secondabsorbance spectra into a composite absorbance spectrum, covering awider wavelength range than either the first or second absorbancespectrum alone. In some embodiments, the device further comprises aheater proximate the sample holder. In some embodiments, the devicefurther comprises a source of moving air proximate the sample holder. Insome embodiments, the device further comprises a mechanism forsequentially presenting samples for analysis by the device. In someembodiments, the device further comprises a dryer positioned to dry asample while a previously-deposited sample is analyzed. In someembodiments, the mechanism for sequentially presenting samples foranalysis by the device comprises a spool holding carrier material thatis incrementally fed across the sample holder.

According to another aspect, a device for measuring absorption ofinfrared radiation by a sample comprises a source of infrared radiation,a sample holder positioned to place a sample in a location to receiveinfrared radiation from the source, and a first Fabry-Perotinterferometer positioned to receive infrared radiation originating fromthe source and passing through the sample. The first Fabry-Perotinterferometer comprises a pair of spaced-apart reflective surfaces, atleast one of which is movable to change the spacing between thereflective surfaces. The device further comprises a first detectorpositioned to receive infrared radiation from the first Fabry-Perotinterferometer and to produce an output signal indicating the intensityof the infrared radiation received at the first detector, and a firstband pass optical filter positioned in an optical path of the devicesuch that infrared radiation from the source passes through the firstband pass optical filter before reaching the first detector, the firstband pass optical filter passing a first band of infrared radiationwavelengths. The device further comprises a second Fabry-Perotinterferometer positioned to receive infrared radiation originating fromthe source and passing through the sample, the second Fabry-Perotinterferometer comprising a pair of spaced-apart reflective surfaces, atleast one of which is movable to change the spacing between thereflective surfaces. The device further comprises a second detectorpositioned to receive infrared radiation from the second Fabry-Perotinterferometer and to produce an output signal indicating the intensityof the infrared radiation received at the second detector, and a secondband pass optical filter positioned in an optical path of the devicesuch that infrared radiation from the source passes through the secondband pass optical filter before reaching the second detector, the secondband pass optical filter passing a second band of infrared radiationwavelengths different from the first. The device further comprises acontroller coupled to the source, the first and second Fabry-Perotinterferometers, and the first and second detectors, the controllerincluding a processor programmed to cause each of the first and secondFabry-Perot interferometers to be tuned to a respective series ofdifferent resonant wavelengths, receive the output signals of therespective detectors at each of the series of resonant wavelengths, andrecord an absorbance spectrum indicating the absorbance of the sample asa function of infrared radiation wavelength within the range ofwavelengths passed by the first and second band pass optical filters. Insome embodiments, the device further comprises a beam splitter thatdirects a first portion of the infrared radiation passing through thesample to the first Fabry-Perot interferometer and passes a secondportion of the infrared radiation passing through the sample to thesecond Fabry-Perot interferometer, such that both the first and secondFabry-Perot interferometers receive infrared radiation from the samplesimultaneously. In some embodiments, the device further comprises anoptical switch that directs infrared radiation passing through thesample to the first Fabry-Perot interferometer when the optical switchis in a first position and directs infrared radiation passing throughthe sample to the second Fabry-Perot interferometer when the opticalswitch is in a second position, such that at most one of the first andsecond Fabry-Perot interferometers receives infrared radiation from thesample at any one time. In some embodiments, the optical switchcomprises a movable mirror. In some embodiments, the device furthercomprises a heater proximate the sample holder. In some embodiments, thedevice further comprises a source of moving air proximate the sampleholder.

According to another aspect, a carrier for holding a sample for proteinquantitation comprises a porous membrane having a number of sampleloading areas designated thereon. The membrane defines a number ofopenings through the membrane, each of the sample loading areas beingsurrounded by openings such that each of the sample loading areas isdefined on a portion of the membrane joined to the remainder of themembrane by bridges of the membrane material. In some embodiments, themembrane material is polyvinylidene difluoride (PVDF),polytetrafluoroethylene (PTFE), or nitrocellulose. In some embodiments,at least the sample areas of the carrier are impregnated with asurfactant.

According to another aspect, a carrier for holding a sample for analysiscomprises a porous membrane having a number of sample loading areasdesignated thereon, and at least the sample areas are impregnated with asurfactant. In some embodiments, the membrane comprises polyvinylidenedifluoride (PVDF), polytetrafluoroethylene (PTFE), or nitrocellulose. Insome embodiments, the entire carrier is impregnated with the surfactant.

According to another aspect, a method of protein quantitation comprisesdirecting infrared radiation to a sample from an infrared radiationsource, at least some of the infrared radiation passing through thesample, and directing infrared radiation having passed through thesample to a band pass optical filter and to the input aperture of aFabry-Perot interferometer. The method further comprises causing theFabry-Perot interferometer to be tuned to a series of different resonantwavelengths, such that filtered infrared radiation emerges from theFabry-Perot interferometer, wherein the filtered infrared radiation ateach tuning includes primarily light in a narrow wavelength band, anddirecting the filtered infrared radiation to a detector. The detectorproduces an output indicating the intensity of the infrared radiationreceived at the detector. The method further comprises receiving thedetector output, and recording an absorbance spectrum indicating theabsorbance of the sample as a function of infrared radiation wavelength.In some embodiments, the band pass filter filters the infrared radiationbefore it enters the Fabry-Perot interferometer. In some embodiments,the band pass filter filters the infrared radiation after it emergesfrom the Fabry-Perot interferometer. In some embodiments, the methodfurther comprises modulating the infrared radiation source at amodulation frequency, and passing the detector output through a lock-inamplifier operating at the modulation frequency. In some embodiments,the band pass filter is a first band pass filter and the absorbancespectrum is a first absorbance spectrum, and the method furthercomprises: replacing the first band pass filter with a second band passfilter, wherein the second band pass filter passes a different set ofinfrared radiation wavelengths than are passed by the first band passfilter; recording a second absorbance spectrum indicating the absorbanceof the sample as a function of infrared radiation wavelength with thesecond band pass filter in place; and combining the first and secondabsorbance spectra into a composite absorbance spectrum. In someembodiments, the method further comprises sequentially presentingsamples to a testing area for quantitation testing using a mechanism. Insome embodiments, the method further comprises drying a sample while aprevious sample is being tested.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic block diagram of a quantitation device inaccordance with embodiments of the invention.

FIG. 2 illustrates a schematic diagram of the operation of a Fabry-Perotinterferometer.

FIG. 3 illustrates the relative transmittance of the interferometer ofFIG. 2 as a function of wavelength for a gap width of 3 microns.

FIG. 4 shows the curve of FIG. 3 with an additional filter curve takenwith a gap width of 2 microns.

FIG. 5 shows a schematic diagram of a quantitation device in accordancewith other embodiments of the invention.

FIG. 6 illustrates a quantitation device in accordance with otherembodiments of the invention.

FIG. 7 illustrates the quantitation device of FIG. 6 with a mirrorremoved from the optical path.

FIG. 8 illustrates a block diagram of a quantitation device inaccordance with another embodiment.

FIG. 9 illustrates a carrier in accordance with embodiments of theinvention, including a number of sample loading areas.

FIG. 10 shows a carrier in accordance with other embodiments of theinvention, including a number of sample loading areas.

FIG. 11 illustrates the use of a heater or moving air source in anembodiment of the invention.

FIG. 12 illustrates a quantitation device in accordance with otherembodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates a schematic block diagram of a quantitation device100 in accordance with embodiments of the invention.

Quantitation device 100 includes a source 101 of infrared (IR) light102. Source 101 is preferably a substantially black body radiatorcontrollable to a temperature that results in sufficient radiationoutput at wavelengths at or near the wavelengths of interest forquantitating the desired analytes. In some embodiments, source 101 maybe a micromachined resistive source of the kind available from AxetrisAG of Kaegiswil, Switzerland. In some embodiments, source 101 may behermetically sealed. Some evidence suggests that a hermetically sealedsource may be more stable than a source that is not hermetically sealed,resulting in better measurement stability. It is believed that the addedstability is due to the protection of the source from air currents thatmight alter the temperature of the source, and therefore the poweroutput of the source. Hermetically sealed IR sources are available fromMicro-Hybrid Electronic GmbH, of Hermsdorf, Germany, and fromHelioworks, Inc., of Santa Rosa, Calif., USA.

A sample holder 103 supports a sample 104, which may be disposed on acarrier 105. Sample holder 103 may be a slot, shelf, aperture, or otherstructure that can support carrier 105. While sample 104 is shown inFIG. 1 as having an appreciable thickness for clarity of illustration,an actual sample may be a very thin spot on a glass carrier, may beimpregnated into a membrane, or may have another form. In any event,sample 104 and carrier 105 are preferably substantially transparent toIR light, except for the absorbing effect of the protein or otheranalyte in the sample.

Carrier 105 may be made of any suitable material, for example glass,silicon, germanium, or another material. In some embodiments, carrier105 may be porous, such as a woven or nonwoven mesh made ofpolyvinylidene difluoride, (PVDF), polytetrafluoroethylene (PTFE), oranother material. In some embodiments, carrier 105 may be coated withone or more surface treatments or impregnated and dried with one or morereagents (e.g. surfactants, detergents, polymers) to make some or all ofthe sample area hydrophobic or hydrophilic, or to impart otherproperties. For example, reagents may impart a shift in the absorbancespectra, cause an increase or decrease in a specific absorbance, orcreate a new absorbance not previously present in the sample absent thereagent.

A lens 106 or other optical system collects IR light that has passedthrough sample 104 and redirects it toward a Fabry-Perot interferometer107. Preferably, a band pass filter 108 is placed in the optical pathbefore the Fabry-Perot interferometer. Filter 108 is shown in FIG. 1 asbeing between lens 106 and Fabry-Perot interferometer 107, but it couldalso be placed differently, for example between source 101 and sample104, or between carrier 105 and lens 106, or between interferometer 107and a detector 109. The purpose of band pass filter 108 will beexplained in more detail below.

While filter 108 is shown as a standalone element in the optical path,it could also be integrated into some other optical component. Forexample, filter 108 may be implemented as a coating on lens 106, orwithin Fabry-Perot interferometer 107, or on another optical element.

The operation of Fabry-Perot interferometer 107 is also explained inmore detail below. Fabry-Perot interferometer 107 acts as a tunablesharp wavelength filter, preferentially passing a narrow band ofwavelengths of the IR light entering it, and substantially blockingother wavelengths. The filtered light exiting Fabry-Perot interferometer107 reaches detector 109, which produces an output signal indicating theintensity of the light reaching detector 109. Fabry-Perot interferometermay be, for example, a micromachined interferometer of the typeavailable from InfraTec GmbH, of Dresden, Germany.

Preferably, detector 109 is placed at the focal point of lens 106.

While lens 106 is shown as a simple single-element lens placed beforefilter 108 in the optical path of the system, other arrangements arepossible. Lens 106 represents any optical system that redirects lightfrom sample 104 toward Fabry-Perot interferometer 107. The opticalsystem may include one or more lens elements, reflectors, or otheroptical components, and maybe place in any workable location in theoptical path, for example after filter 108.

A controller 110 is coupled to IR source 101, Fabry-Perot interferometer107, and detector 109, for controlling the operation of the system,storing measurement information, presenting results, and otherfunctions. Controller 110 causes Fabry-Perot interferometer 107 to betuned to a series of different resonant wavelengths, and measures theoutput of detector 109 at each wavelength. Using this data, controller110 can construct an absorbance spectrum of the sample. For the purposesof this disclosure, the terms “absorbance spectrum” and “absorptionspectrum” are used interchangeably. This spectrum can be used toquantify the amount of analyte in the sample. Typically, the absorptionspectrum is analyzed to estimate the size of any absorption feature ofinterest that is associated with the material being quantified. Theabsorption feature manifests itself as a loss in power when compared toa measurement of a sample without the material of interest. Typically, areference scan is done on the sample substrate without the material ofinterest so this can be directly compared to measurements done with thematerial of interest. In one embodiment, the measurement of the materialof interest is divided by the reference measurement, which will give thefraction of light loss due to the material of interest. Another way toexplain the ratio would be the transmittance through the material ofinterest. By taking the measurement at many wavelengths around theabsorption feature of interest, a transmittance spectra can be plottedwhich shows a dip around any absorption features. The magnitude of theabsorption feature can be calculated from the plot by various means,such as looking at areas of the curve with no absorption feature andestimating the height of the absorption feature relative to areas withno absorption.

In some embodiments, detector 109 may be a pyroelectric detector. Apyroelectric detector includes a material that generates an electricvoltage as the material is heated or cooled—a phenomenon called thepyroelectric effect. Suitable pyroelectric detectors are also availablefrom InfraTec GmbH.

Because the pyroelectric effect requires the detecting material to beheated and cooled, source 101 may be pulsed or modulated as shown at 111if a pyroelectric detector is used. In some embodiments, source 101 maybe modulated at about 10 Hz. Because source 101 is micromachined, itscomponents are very small and are able to heat and cool very rapidly,enabling the modulation of source 101.

The modulation of source 101 has the additional benefit that lock-indetection may be used to read the outputs of detector 109. Lock-indetection is a technique for greatly reducing the effect of noise bymultiplying a signal to be measured by a reference signal of the samefrequency, and integrating the result. Components of the signal to bemeasured that are not at the measurement and reference frequency aregreatly attenuated, enabling extraction of measurement data fromextremely noisy signals. A lock-in amplifier 112 may be placed, forexample, in controller 110 or another location in the system.Modulation, lock-in detection, or both may be used in any embodimentdescribed herein.

In addition to the performance benefits resulting from modulation, whichis enabled by the small size of a micromachined source, the use of amicromachined source 101 and a micromachined interferometer 107 meansthat quantitation device 100 can be made very compact as compared withprior devices, and at dramatically lower cost.

The use of a Fabry-Perot interferometer for protein quantitation is asomewhat surprising discovery, as this kind of interferometer is knownto have a narrower wavelength measurement range than, for example, aMichelson interferometer. The narrowness of the measurement range can beunderstood as follows.

FIG. 2 shows a schematic diagram of the operation of Fabry-Perotinterferometer 107. Fabry-Perot interferometer 107 includes two flatpartially transmitting mirrors 201, spaced apart by a distance D, andhaving a reflectance R. Entering light 202 reaches the gap betweenmirrors 201 and undergoes multiple reflections between mirrors 201before exiting through an exit aperture 203 of interferometer 107. Lighthaving a wavelength of twice the spacing D or any submultiple thereof(D, D/2, D/3, etc.) “resonates” within the gap, and undergoesconstructive interference. Light of other wavelengths undergoes somelevel of destructive interference. The net effect is that interferometer107 is a sharp wavelength filter, passing light having a resonantwavelength much more readily than light of other wavelengths. Exitinglight 204 is therefore composed predominantly of the resonantwavelengths.

Distance D is adjustable under the control of controller 110, throughcontrol electrodes 205.

The filter characteristic of interferometer 107 is described by the Airyformula:

$T = {\left( {1 - \frac{A}{\left( {1 - R} \right)}} \right)^{2}\frac{1}{1 + {\frac{4R}{\left( {1 - R} \right)^{2}}{\sin^{2}\left( {{2\pi\;{nD}\frac{1}{\lambda}\cos\;\beta} - \varphi} \right)}}}}$where:

-   -   T is the transmittance of the filter as a function of        wavelength;    -   n is the refractive index of the gap material (˜1.0 for air);    -   D is the gap width;    -   β is the angle of incidence;    -   R is the mirror reflectance;    -   A is the mirror absorptance; and    -   φ is the phase at reflection        (See Ebermann, Martin, et al. “Design; Operation and Performance        of a Fabry-Perot-Based MWIP, Microspectrometer.” Sensor+ Test        Confrence. Vol. 2009. 2009.)

FIG. 3 illustrates the relative transmittance of interferometer 107 as afunction of wavelength for a gap width D of 3 microns (with n=1, β=0,R=0.75, A=0.02, and φ=0). As is apparent, the transmittance exhibitssharp peaks at 6 microns and submultiples thereof—that is at 3, 2, 1.5,1.2, and 1 microns. Interferometer 107 thus acts as a sharp wavelengthfilter, passing wavelengths near the peaks and substantially blockingother wavelengths.

If no other filtering were done, the detector 109 would receive light atall of the peak wavelengths, and it would not be possible to determinean accurate absorbance spectrum for sample 104 based on the output ofdetector 109 because each reading would include signal resulting frommultiple wavelength bands. However, filter 108 may be configured toblock the shorter peak wavelengths, as shown by the (idealized) shadedarea of FIG. 3, leaving only the rightmost peak to be passed. Forexample, filter 108 may be dichroic filter with a cutoff wavelength ofjust over 3 microns (in this example), so that wavelengths longer thanthe cutoff are passed while wavelengths shorter than the cutoff areblocked. The position of rightmost peak 301 varies with the gap width D.

For example, FIG. 4 shows the curve of FIG. 3 with an additional filtercurve taken with a gap width D of 2 microns, and shown in dashed lines.In the dashed curve, the rightmost peak has shifted to the left (to ashorter wavelength). That is, interferometer 107 has been tuned to aresonant wavelength of 4 microns. As can be seen, interferometer 107 hasa limited workable range of wavelengths for detecting an absorbancespectrum. If the mirror spacing D is made too small, the rightmost peakwill be shifted to a wavelength short enough to be blocked by filter108. If the mirror spacing D is made too large, then secondary peak 401will move to the right and fall outside the filtration band of filter108. In that case, detector 109 would receive light from both of thefirst two peaks, and it would be impossible to determine the absorbanceat a single specific wavelength. The workable range extends roughly fromthe cutoff wavelength of filter 108 to double the cutoff wavelength. Theworkable range is also sometimes called the free spectral range.

This limitation on the workable wavelength range of a Fabry-Perotinterferometer would seem to weigh against the use of a Fabry-Perotinterferometer for quantitation of proteins and other analytes, becausedifferent analytes may absorb at different wavelengths which may bewidely different from each other.

However, embodiments of the invention enable use of a Fabry-Perotinterferometer for quantitation.

In one embodiment, the cutoff wavelength of filter 108 is chosen so thatthe workable range of interferometer 107 encompasses the absorbancewavelengths of one or more analytes of interest. For example, manyproteins absorb at wavelengths corresponding to bending and stretchingvibrations in certain chemical bonds in the proteins. Two bands ofparticular interest are the “Amide I” and “Amide II” bands. The Amide Iband results primarily from stretching vibrations in C═O and C—N bonds,and falls between about 5.88-6.25 microns (wave number 1600-1700 cm⁻¹).The Amide II band results primarily from bending vibrations in N—Hbonds, and falls between about 6.33-6.62 microns (wave number 1510-1580cm⁻¹).

By properly selecting the cutoff wavelength of filter 108, the workablerange of interferometer 107 can be made to encompass both the Amide Iand Amide II bands, making quantitation device 100 useful for manyprotein quantitation experiments. For example, a cutoff wavelengthbetween about 3.5 and 5 microns may be used. In one preferred embodimentparticularly suitable for protein quantitation, filter 108 has a cutoffwavelength of just over 4.0 microns, giving the device a workingwavelength range of about 5 to 8 microns.

Similarly, DNA and RNA are known to absorb at specific IR wavelengths,for example at about 8 microns.

FIG. 5 shows a schematic diagram of a quantitation device 500 inaccordance with other embodiments of the invention. Example quantitationdevice 500 can have an extended measurement range as compared withdevice 100 discussed above. This is accomplished by including twoFabry-Perot interferometers with different measurement ranges within thesame device 500.

In example quantitation device 500, a source 501 produces IR light 502to illuminate a sample 504. Source 501 may be similar to source 101discussed above, and is preferably a substantially black body radiatorcontrollable to a temperature that results in sufficient radiationoutput at wavelengths at or near the wavelengths of interest forquantitating the desired analytes.

A sample holder 503 supports the sample 504, which may be disposed on acarrier 505. While sample 504 is shown in FIG. 5 as having anappreciable thickness for clarity of illustration, an actual sample maybe a very thin spot on a glass carrier, may be impregnated into amembrane, or may have another form. In any event, sample 504 and carrier505 are preferably substantially transparent to IR light, except for theabsorbing effect of the protein or other analyte in the sample.

A lens 506 or other optical system collects IR light that has passedthrough sample 504 and redirects it toward a beam splitter 511. Beamsplitter 511 may include, for example, a partially silvered mirror thatdivides the light coming from lens 506 and directs the divided portionsin different directions. In FIG. 5, one portion (for example 50% oranother portion) of the light is directed to Fabry-Perot interferometer507 a. Another portion of the light is directed to a second Fabry-Perotinterferometer 507 b. Other optical elements may be present forfacilitating the direction of light to the two interferometers, forexample mirrors such as mirror 512, collimators, and the like. In otherembodiments, the multiple interferometers may be spatially arranged tosimplify the optical arrangements. For example, second interferometer507 b could be placed with its axis parallel to beam 513, so that mirror512 would not be needed.

While beam splitter 511 is shown as being placed after lens 506 in theoptical path, it may also be placed before lens 506. In someembodiments, lens 506 provides collimation of the light, to help avoiddegradation of the intensity of the light as it travels to the twointerferometers, which may be different optical distances from sample504. In other embodiments, the system may be constructed so that thepath lengths from sample 504 to the two interferometers are the same.Collimation may be used in any event.

A first filter 508 a is placed in the optical path before firstinterferometer 507 a. Filter 508 a is a band pass filter, having a firstcutoff wavelength, setting the working wavelength range of firstinterferometer 507 a. By way only of example, first filter 508 a mayhave a cutoff wavelength of about 3 microns, blocking all wavelengthsshorter than 3 microns and passing all wavelengths longer than 3microns, so that the working wavelength range of interferometer 507 a isabout 3 to 6 microns.

A second filter 508 b is placed in the optical path before secondinterferometer 507 b. Filter 508 b is also a band pass filter having adifferent cutoff wavelength than first filter 508 a. By way only ofexample, second filter 508 b may have a cutoff wavelength of about 5microns, blocking all wavelengths shorter than 5 microns and passing allwavelengths longer than 5 microns, so that the working wavelength rangeof interferometer 507 b is about 5 to 10 microns.

Detectors 509 a and 509 b receive the light passed by interferometers507 a and 507 b respectively, and produce signals indicating theintensity of the light received by detectors 509 a and 509 b. Acontroller 510 is coupled to source 501, interferometers 507 a and 507b, and detectors 509 a and 509 b, and is configured to control theoperation of the system and to receive signals from detectors 509 a and509 b.

In this arrangement, interferometers 507 a and 507 b can essentially beoperated in parallel, to test the same sample 504 across two wavelengthbands. The workable wavelength range of quantitation device 500 spansthe combined ranges of interferometers 507 a and 507 b, enabling testingof a wider variety of analytes as compared with quantitation device 100.In the above example, the wavelength ranges of interferometers 507 a and507 b overlap, and combine for a working wavelength range of about 3 to10 microns. In other embodiments, different ranges may be selected. Theranges of the multiple interferometers may overlap, may abut, or may beseparated by gaps so that absorption data is not gathered for somewavelengths. Controller 510 reads the outputs of detectors 509 a and 509b as interferometers 507 a and 507 b are moved across their desiredranges, and then combines the readings into a composite absorptionspectrum. Interferometers 507 a and 507 b may be operated and sensed oneat a time in sequence, or may be operated and sensed in parallel, withcontroller 510 reading the outputs of detectors 509 a and 509 balternately or in another interleaved pattern.

In one embodiment, the wavelength ranges of the two interferometers maybe selected so that device 500 can detect proteins in wavelengths ofabout 5.8-6.3 microns with one of the interferometers and lipids inwavelengths of about 3.3-3.6 microns with the other interferometer.

In other embodiments, more than two interferometers may be provided,each in conjunction with a band pass filter having a different cutoffwavelength, further extending the workable range of the overallinstrument. The workable wavelength ranges of the interferometers in amultiple-interferometer instrument may overlap, may abut, or may beseparated by gaps in which certain wavelengths are not sensed.

The arrangement of FIG. 5 has the advantage that interferometers 507 aand 507 b can be operated in parallel, enabling measurement over a widerange of wavelengths in a way that may appear nearly simultaneous.However, this arrangement may have the disadvantage that the lightintensity reaching at least one of the interferometers is half or lessthe intensity that would be present if beam splitter 511 were notpresent, resulting in less measurement signal and possibly a worsesignal-to-noise ratio in the outputs of detectors 509 a and 509 b. Ifthe light is divided among more than two interferometers, then theintensity would be further reduced.

FIG. 6 illustrates a quantitation device 600 in accordance with otherembodiments of the invention. In device 600, a movable mirror 601 isused instead of beam splitter 511. Movable mirror 601 is an example ofan optical switch. The other components in FIG. 6 are similar to thosein FIG. 5, and are given the same reference numerals. When mirror 601 isin place as shown, substantially all light from lens 506 is directed tointerferometer 507 b. FIG. 7 shows quantitation device 600 with mirror601 removed from the optical path, so that the light from lens 506 isdirected to interferometer 507 a. In device 600, interferometers 507 aand 507 b are preferably used one at a time. That is, one of theinterferometers is used to completely measure its wavelength range,mirror 601 is moved, and then the other interferometer is used tomeasure its wavelength range. While the measurements are sequential, thefull intensity of light coming from lens 506 is available for bothmeasurements.

In FIG. 7, mirror 601 is shown as having been translated out of theoptical path. In other embodiments, a rotational motion may be used, ora combination of rotation and translation. In other embodiments, mirror601 may be an electronically controllable reflector, and may be madereflective or transmissive as needed rather than being moved. Othermethods of switching light from one interferometer to another may beenvisioned and used as well. More than two interferometers may be used,in conjunction with other optical arrangements that switch the lightamong any workable number of interferometers.

FIG. 8 illustrates a block diagram of a quantitation device 800 inaccordance with another embodiment. Example quantitation device 800 issimilar in many respects to quantitation device 100 shown in FIG. 1, andsimilar elements are given the same reference numerals. However, ratherthan a single filter, quantitation device 800 includes twointerchangeable filters 801 and 802, having different cutoffwavelengths. Because the workable or free spectral range of Fabry-Perotinterferometer 107 is fundamentally determined by the cutoff wavelengthof the filter used with it, the workable or free spectral range ofdevice 800 can be modified by swapping filters 801 and 802. When filter801 is placed in the optical path, the workable range is determined bythe cutoff wavelength of filter 801. When filter 802 is placed in theoptical path, the workable range is determined by the cutoff wavelengthof filter 802. By switching filters 801 and 802 into the optical path atdifferent times, the working range of device 800 can be the combinedworking ranges determined by the two filters. This is in much the sameway that the working range of device 600 described above includes theranges of two interferometers used one at a time, except that device 800accomplishes the range extension using only one interferometer.Presuming that a suitable interferometer is used, for example withsufficient mirror travel, device 800 has the advantage that the cost ofa second interferometer may be saved. As in other embodiments, theranges determined by filters 801 and 802 may overlap, may abut, or maybe separated so that some wavelengths are not covered in the compositeworking range. More than two filters may be provided.

According to another aspect, a sample carrier such as carrier 105 may beformed in such a way as to facilitate loading of samples into thequantitation device.

FIG. 9 shows a carrier 901 in accordance with embodiments of theinvention, including a number of sample loading areas 902. Carrier 901may be, for example, a porous membrane made from PVDF, PTFE,nitrocellulose, or another suitable material. Border markings 903 may beprinted around loading areas 902 to guide a user in where to placesamples for testing. Carrier 901 may then be placed into a quantitationdevice such as device 100, and the different loading areas 902 may besequentially moved into the optical path of the device for testing. Themovement may be done manually by the user, or the device may include amotorized mechanism for moving carrier 901.

In some embodiments, border markings may serve other functions as well.For example, border markings 903 may be made hydrophobic, to preventliquid samples placed in sample loading areas 902 from migrating out ofthe loading area. The interior of sample loading areas 902 could also betreated to be hydrophilic, to further assist in containing sampleliquids. In other embodiments, annular regions of the membranesurrounding the sample loading areas may be compressed to preventmigration of the sample out of the sample loading areas.

FIG. 10 shows a carrier 1001 in accordance with other embodiments of theinvention, including a number of sample loading areas 1002. Carrier 1001may also be, for example, a porous membrane made from PVDF, PTFE,nitrocellulose, or another suitable material. Openings 1003 are cut intocarrier 1001, surrounding each of the sample loading areas 1002.Openings 1003 serve to substantially prevent liquid from the sampleareas from migrating out of the sample areas. Bridges 1004 supportsample loading areas 1002. Bridges 1004 are preferably narrow enoughthat any migration of liquid along them is inconsequential. In someembodiments, for example when carrier 1001 is made of a porous membrane,bridges 1004 may be treated with a solidifying substance such as anadhesive to block any migration of liquid along them. In someembodiments, the bridges may be compressed to block migration of liquid.

It will be recognized that single-sample carriers are possible, as arecarriers having sample areas arranged differently than in FIGS. 9 and10. For example, a disk-shaped carrier may include sample areas in acircular pattern, and the sample areas may be presented to the opticalpath of the quantitation device using a manual or automatic rotatingmechanism. In other embodiments, a carrier may include a two-dimensionalarray of sample areas, or another arrangement.

According to another aspect, measures are taken to prepare the sample tobe measured to provide for robust measurement.

For example, water absorbs infrared radiation heavily, so any sample tobe measured with a device embodying the invention must be thoroughlydried. In some embodiments, a quantitation device may include featuresfor promoting the drying of a sample. For example, FIG. 11 illustratesthe use of a heater 1101 or moving air source 1102 in an embodiment ofthe invention. Heater 1101 and air source 1102 are shown in the contextof quantitation device 100, but such features may be present in anyembodiment. Only one of heater 1101 or air source 1102 may be present,or both may be present. Heater 1101 may be a resistive heater placednear the sample holder, such as immediately over or under the sample,possibly surrounding the sample, so that heat may be temporarily appliedto ensure full drying of the sample. Other kinds of heaters may be used.In other embodiments, a source of flowing air may be provided near thesample holder and aimed at the sample, to promote drying of the sample.Flowing air source 1102 may be a rotary or piezoelectric fan, or anotherkind of air source. Heat and moving air may be used individually or incombination. Preferably, any heater or air source can be switched on andoff as needed, for example under the control of a controller such ascontroller 110.

FIG. 12 illustrates a quantitation device 1200 in accordance with otherembodiments of the invention. Several elements of device 1200 aresimilar to elements of device 100, and are given the same referencenumbers. Quantitation device 1200 includes an additional capability forpresenting samples for analysis to the device. In device 1200, samplesare deposited on a strip 1201 of carrier material, which may be similarto the material of carrier 105, 901, or 1001. Sample loading areas 1202may be indicated on strip 1201, and may be isolated by bridges from therest of strip 1201, similar to the isolation of sample loading areas1002 on carrier 1001 described above. Strip 1201 is provided on a supplyspool 1203, and is threaded across sample holder 103 to a take up spool1204.

This arrangement allows for efficient usage of the device. Samples maybe placed on strip 1201 and incrementally advanced toward sample holder103. While one sample, is being characterized by Fabry-Perotinterferometer 107, subsequent samples may be dried, for example by amoving air source 1102, a heating element (not shown), or other dryingdevices or combinations of devices. Supply and take up spools 1203 and1204 may be motorized and controlled by controller 110, may be manuallycontrolled, or may be controlled in some other way.

Other mechanisms for sequentially presenting samples for analysis may beused in other embodiments. For example, sample areas could be designatedon a flat rotating disk, in a rectangular array, or in otherarrangements.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. The invention has now beendescribed in detail for the purposes of clarity and understanding.However, those skilled in the art will appreciate that certain changesand modifications may be practiced within the scope of the appendedclaims.

What is claimed is:
 1. A device for measuring absorption of light by asample, the device comprising: a source of infrared radiation; a sampleholder positioned to place a sample in a location to receive infraredradiation from the source; a Fabry-Perot interferometer positioned toreceive infrared radiation originating from the source and passingthrough the sample, the Fabry-Perot interferometer comprising a pair ofspaced-apart reflective surfaces, at least one of which is movable tochange the spacing between the reflective surfaces; a detectorpositioned to receive infrared radiation from the Fabry-Perotinterferometer and to produce an output signal indicating the intensityof the infrared radiation received at the detector; a first band passoptical filter positioned in an optical path of the device such thatinfrared radiation in a first wavelength band from the source passesthrough the first band pass optical filter before reaching the detector;a second band pass optical filter that passes infrared radiation in asecond wavelength band different from the first wavelength band; amechanism for removing the first band pass optical filter from theoptical path and placing the second band pass filter in the optical pathsuch that infrared radiation from the source passes through the secondband pass optical filter before reaching the detector; and a controllercoupled to the source, the Fabry-Perot interferometer, and the detector,the controller including a processor programmed to: cause theFabry-Perot interferometer to be tuned to a series of different resonantwavelengths with the first band pass optical filter in place in theoptical path; receive the output signal of the detector at each of theseries of resonant wavelengths; and record an absorbance spectrumindicating the absorbance of the sample as a function of infraredradiation wavelength.
 2. The device of claim 1, wherein the source ofinfrared radiation is a micromachined resistive source.
 3. The device ofclaim 1, wherein the source of infrared radiation is hermeticallysealed.
 4. The device of claim 1, wherein the Fabry-Perot interferometeris a micromachined Fabry-Perot interferometer.
 5. The device of claim 1,further comprising an optical system positioned to direct infraredradiation passing through the sample toward an entrance aperture of theFabry-Perot interferometer.
 6. The device of claim 1, wherein thedetector is a pyroelectric detector.
 7. The device of claim 1, whereinthe source is modulated at a predetermined measurement frequency.
 8. Thedevice of claim 7, further comprising a lock-in amplifier that receivesthe signal from the detector, enabling lock-in detection of the infraredradiation intensity.
 9. The device of claim 1, wherein the series ofwavelengths is a first series of wavelengths and the absorbance spectrumis a first absorbance spectrum, and wherein the processor of thecontroller is further programmed to: cause the Fabry-Perotinterferometer to be tuned to a second series of different resonantwavelengths with the second band pass optical filter in place in theoptical path; receive the output signal of the detector at each of thesecond series of resonant wavelengths; and record a second absorbancespectrum indicating the absorbance of the sample as a function ofinfrared radiation wavelength within the range of wavelengths passed bythe second band pass optical filter.
 10. The device of claim 9, whereinthe processor of the controller is further programmed to combine thefirst and second absorbance spectra into a composite absorbancespectrum, covering a wider wavelength range than either the first orsecond absorbance spectrum alone.
 11. A device for measuring absorptionof infrared radiation by a sample, the device comprising: a source ofinfrared radiation; a sample holder positioned to place a sample in alocation to receive infrared radiation from the source; a firstFabry-Perot interferometer positioned to receive infrared radiationoriginating from the source and passing through the sample, the firstFabry-Perot interferometer comprising a pair of spaced-apart reflectivesurfaces, at least one of which is movable to change the spacing betweenthe reflective surfaces; a first detector positioned to receive infraredradiation from the first Fabry-Perot interferometer and to produce anoutput signal indicating the intensity of the infrared radiationreceived at the first detector; a first band pass optical filterpositioned in an optical path of the device such that infrared radiationfrom the source passes through the first band pass optical filter beforereaching the first detector, the first band pass optical filter passinga first band of infrared radiation wavelengths; a second Fabry-Perotinterferometer positioned to receive infrared radiation originating fromthe source and passing through the sample, the second Fabry-Perotinterferometer comprising a pair of spaced-apart reflective surfaces, atleast one of which is movable to change the spacing between thereflective surfaces; a second detector positioned to receive infraredradiation from the second Fabry-Perot interferometer and to produce anoutput signal indicating the intensity of the infrared radiationreceived at the second detector; a second band pass optical filterpositioned in an optical path of the device such that infrared radiationfrom the source passes through the second band pass optical filterbefore reaching the second detector, the second band pass optical filterpassing a second band of infrared radiation wavelengths different fromthe first; and a controller coupled to the source, the first and secondFabry-Perot interferometers, and the first and second detectors, thecontroller including a processor programmed to: cause each of the firstand second Fabry-Perot interferometers to be tuned to a respectiveseries of different resonant wavelengths; receive the output signals ofthe respective detectors at each of the series of resonant wavelengths;and record an absorbance spectrum indicating the absorbance of thesample as a function of infrared radiation wavelength within the rangeof wavelengths passed by the first and second band pass optical filters.12. The device of claim 11, further comprising a beam splitter thatdirects a first portion of the infrared radiation passing through thesample to the first Fabry-Perot interferometer and passes a secondportion of the infrared radiation passing through the sample to thesecond Fabry-Perot interferometer, such that both the first and secondFabry-Perot interferometers receive infrared radiation from the samplesimultaneously.
 13. The device of claim 11, further comprising anoptical switch that directs infrared radiation passing through thesample to the first Fabry-Perot interferometer when the optical switchis in a first position and directs infrared radiation passing throughthe sample to the second Fabry-Perot interferometer when the opticalswitch is in a second position, such that at most one of the first andsecond Fabry-Perot interferometers receives infrared radiation from thesample at any one time.
 14. The device of claim 13, wherein the opticalswitch comprises a movable mirror.
 15. The device of claim 11, furthercomprising a heater proximate the sample holder.
 16. A method of proteinquantitation, the method comprising: directing infrared radiation to asample from an infrared radiation source, at least some of the infraredradiation passing through the sample; directing infrared radiationhaving passed through the sample to a first band pass optical filter andto an input aperture of a Fabry-Perot interferometer; causing theFabry-Perot interferometer to be tuned to a series of different resonantwavelengths, such that filtered infrared radiation emerges from theFabry-Perot interferometer, wherein the filtered infrared radiation ateach tuning includes primarily light in a narrow wavelength band;directing the filtered infrared radiation to a detector, the detectorproducing an output indicating the intensity of the infrared radiationreceived at the detector; receiving the detector output; recording afirst absorbance spectrum indicating the absorbance of the sample as afunction of infrared radiation wavelength; replacing the first band passfilter with a second band pass filter, wherein the second band passfilter passes a different set of infrared radiation wavelengths than arepassed by the first band pass filter; recording a second absorbancespectrum indicating the absorbance of the sample as a function ofinfrared radiation wavelength with the second band pass filter in place;and combining the first and second absorbance spectra into a compositeabsorbance spectrum.
 17. The method of claim 16, further comprising:modulating the infrared radiation source at a modulation frequency; andpassing the detector output through a lock-in amplifier operating at themodulation frequency.
 18. The method of claim 16, further comprisingsequentially presenting samples to a testing area for quantitationtesting using a mechanism.